WO2004011599A2 - Polypeptide cleavage process - Google Patents

Polypeptide cleavage process Download PDF

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Publication number
WO2004011599A2
WO2004011599A2 PCT/US2003/016647 US0316647W WO2004011599A2 WO 2004011599 A2 WO2004011599 A2 WO 2004011599A2 US 0316647 W US0316647 W US 0316647W WO 2004011599 A2 WO2004011599 A2 WO 2004011599A2
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WO
WIPO (PCT)
Prior art keywords
peptide
palladium
cleavage
chimeric protein
cys
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PCT/US2003/016647
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English (en)
French (fr)
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WO2004011599A3 (en
Inventor
Jin Seog Seo
Barton Holmquist
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Restoragen, Inc.
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Application filed by Restoragen, Inc. filed Critical Restoragen, Inc.
Priority to DE60334912T priority Critical patent/DE60334912D1/de
Priority to AT03771535T priority patent/ATE487486T1/de
Priority to EP03771535A priority patent/EP1531836B1/de
Priority to AU2003276835A priority patent/AU2003276835A1/en
Priority to CA2485959A priority patent/CA2485959C/en
Publication of WO2004011599A2 publication Critical patent/WO2004011599A2/en
Publication of WO2004011599A3 publication Critical patent/WO2004011599A3/en
Priority to US10/997,822 priority patent/US7592432B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/60—Growth hormone-releasing factor [GH-RF], i.e. somatoliberin

Definitions

  • the invention provides highly specific, conformationally independent, palladate promoted hydrolytic cleavage of polypeptides, including relatively insoluble chimeric proteins in the form of inclusion bodies.
  • Cleavage from the leader sequence may be accomplished by placing a sequence of amino acids at the junction of the leader and the peptide which are specifically recognized and cleaved under appropriate conditions, e.g. acid cleavage or enzymatic cleavage.
  • acid cleavage or enzymatic cleavage For example, introduction of acid-labile aspartyl-proline linkage between the two segments of a chimeric protein facilitates cleavage at low pH. This technique does not work if the product peptide, which is to be cleaved from the polypeptide, is not acid-labile.
  • Chimeric proteins comprising hormones such as insulin and somatostatin have been cleaved with cyanogen bromide, which is specific for the carboxyl side of methionine residues. This method is not suitable when the product peptide contains methionine residues.
  • Chimeric proteins containing a chicken pro-alpha-2 collagen linker could be specifically degraded by purified microbial collagenase to release the components of the chimeric protein.
  • Use of proteolytic enzymes is expensive, product peptide cleavage yield is frequently low, and it can prove difficult to separate the enzyme from a desired peptide product.
  • Other methods for purification and recovery of a desired recombinant protein include construction of a poly-arginine tail at the C-terminus of the polypeptide. The arginine residues increase the overall basicity of the protein, which facilitates purification by ion exchange chromatography. Subsequent removal of the poly-arginine tail by carboxypeptidase B regenerates the desired protein and allows purification from basic contaminants due to the reduction in pi of the desired protein.
  • Acid cleavage can be accomplished by placing a specific dipeptide at the junction of the leader sequence and the peptide. Selection of the second amino acid will determine the rate at which the dipeptide bond is cleaved under acidic conditions. Of course, if the desired peptide contains any internal dipeptide sequences that are acid cleavable, then the cleavage site at the junction of the leader and the peptide must undergo acid cleavage at a substantially greater rate than the internal cleavage in order to avoid unacceptable loss of yield.
  • Zhu et al. states at p. 5220 that 100 mM HBF 4 , HClO 4 , and CF 3 COOH, or 70 % formic acid, cleaved cytochrome-c.
  • the reference thereafter concludes that cleavage was inhibited by the presence of chloride ions, a notable drawback as proteins purified from biological systems will almost invariably contain chloride ions.
  • Zhu et al. at p. 5219 conclude that the rate of hydrolysis depended on conformational aspects of cytochrome c (i.e., cleavage was thought to be affected by the size of the cleaved peptide fragment and hence, the sequence of the polypeptide to be cleaved).
  • HBF 4 only the HBF 4 reaction media cleaved the chimeric protein at the experimental conditions of 40 ° and reaction time of 48 hours.
  • the cleavage in HBF 4 was said to be pH-dependent or independent depending on the amino acid adjacent to Cys-His: cleavage at Cys-His-Lys was pH-independent while cleavage at Cys-His-Arg was pH-dependent. Id. Further, cleavage at either site in HBF 4 was temperature dependent; when the temperature was increased to 60 °C the chimeric protem solubihzed and it was no longer possible to cleave selectively. Id. Dou, et al.
  • the process of the instant invention provides a highly site-specific process for palladium-promoted hydrolytic cleavage of polypeptides under reaction conditions that are relatively insensitive to variations in reactant concentration, temperature or pH.
  • the process is conformationally and sequence- independent, i.e., it achieves high cleavage yield irrespective of the type of amino acid groups adjacent to the specified cleavage site.
  • the process of the instant invention cleaves polypeptides under conditions which limit the formation of unwanted side- products and which enable the use of chloride-containing catalysts and reaction- media.
  • the process may be employed to cleave, with a high degree of specificity, a single-copy recombinant polypeptide, a multi-copy recombinant polypeptide or a single or multi-copy recombinant chimeric protein construct. Consequently, the process can produce numerous cleaved peptide fragments suitable for further processing. More specifically, the invention provides a process for cleaving a polypeptide at a Cys-His cleavage site in a reaction medium comprising concentrated formic acid and a palladate promotor.
  • a chimeric protein comprised of a leader sequence joined by a Cys-His cleavage site to the N-te ⁇ ninus of the peptide is cleaved by solubilizing the chimeric protein in a reaction mixture comprised of a palladium promotor dissolved in a high- concentration formic acid, wherein the concentration of the formic acid in the reaction mixture is between about 1 to about 22 molar.
  • reaction media solubilize chimeric proteins or inclusion bodies previously considered to be relatively insoluble and such solubilization, rather than decreasing the specificity of cleavage, actually leads to improved yields of cleaved peptide.
  • the process cleaves such chimeric proteins in a manner that facilitates additional processing necessary to post-translationally modify the cleaved peptide, e.g., amidation.
  • the invention provides a process for preparing a peptide by cleaving a chimeric protein which is comprised of a leader sequence joined by a Cys-His cleavage site to the N-terminus of the peptide by solubilizing and reacting the protein in a reaction mixture comprising a high concentration of formic acid solvent and a palladium promotor, wherein the concentration of the formic acid in the reaction mixture is between about 13 to about 22 molar.
  • the molar ratio of palladium promotor to chimeric protein in the reaction mixture is from about 2:1 to about 20:1,
  • the reaction mixture temperature is maintained at about 50 °C to about 70 °C; and
  • the peptide is recovered from the reaction mixture in a substantially formylated form which is deformylated in a second hydrolytic step removing formic acid from the cleavage reaction product by reacting the cleaved and formylated peptide with a nucleophile such as 150 mM glycine, or 25 mM hydroxylamine, at a pH in the range of 6 to 9 or by incubating the formylated product in dilute acid for 4 to 16 hours.
  • a nucleophile such as 150 mM glycine, or 25 mM hydroxylamine
  • Palladium promoted acid cleavage in accordance with the process of the instant invention is facilitated by the use of these high concentrations of formic acid which solubilizes inclusion bodies such as T7tag-Vg-D 4 K-CH-GRF(l-44)- Ala (SEQ LD NO:2), T7tag-Vg-D 4 K-CH-GRF(l-44)-CH (SEQ LD NO:6) or T7tag-Vg-G 5 PR-CH-PTH(l-34) (SEQ ID NO:4).
  • FIGURE 1 illustrates the chemical structure of various palladium complexes useful in the process of the instant invention.
  • FIGURE 2 illustrates the reactivity of the precursor peptide T7tag-Vg- D 4 K-CH-GRF(l-44)-CH (SEQ LD NO:6) at 60°C as a function of palladate and formic acid concentration.
  • FIGURE 3 illustrates the cleavage of T7tag-Vg-D 4 K-CH-GRF(l-44)-CH
  • FIGURE 4 illustrates the nucleotide (SEQ ID NO: 1) and amino acid (SEQ LD NO:2) sequences of the chimeric protein T7tag-Vg-D 4 K-CH-GRF(1- 44)-Ala
  • FIGURE 5 illustrates the nucleotide (SEQ ID NO:3) and amino acid
  • FIGURE 6 illustrates the nucleotide (SEQ ID NO: 5) and amino acid (SEQ ID NO:6) sequences of the chimeric protein T7tag-Vg-D 4 K-CH-GRF(1- 44)-CH.
  • process of the instant invention may be employed to cleave naturally occurring peptides, synthetically derived peptides, and recombinantly derived peptides.
  • the embodiments described in detail hereinafter relate to cleavage of recombinantly expressed chimeric proteins in the form of inclusion bodies.
  • the process of the instant invention can be applied to cleave chimeric proteins which have been recombinantly expressed in a microbial host cell using known techniques of recombinant DNA production and which have been recovered from the host cell in the form of an inclusion body.
  • Any suitable host cell known to be useful for the expression of proteins by recombinant DNA methods may be employed to express such chimeric proteins, including prokaryotic and eukaryotic host cells and cell lines.
  • E. coli is a preferred host cell.
  • the host cell contains an expression vector which encodes the chimeric protein under the control of a regulatory sequence which is capable of directing its expression in the host, as well as an origin of replication that is functional in the host cell.
  • the vector may contain other DNA sequences conventionally employed in recombinant DNA technology such as sequences encoding selectable markers.
  • Methods for expressing a foreign gene in a host organism also are well known in the art (see, e.g., Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2 nd ed., 1989).
  • the gene encoding a particular polypeptide can be constructed by chemically synthesizing the entire nucleotide sequence, by amplification, such as by the polymerase chain reaction (PCR), or by cloning the gene of interest. The gene is then subcloned into an appropriate expression vector.
  • PCR polymerase chain reaction
  • Cloning vectors, expression vectors, plasmids, and viral vectors are well known in the art (see, e.g., Maniatis et al., supra, and Goedell, Methods in ⁇ nzymology, Vol. 185 (Academic Press 1990)).
  • Example 1 provides a description of the preparation of a T7-based expression system useful for high-level expression of mammalian proteins in E. coli.
  • the host cell containing the expression vector is grown and the chimeric protein expressed under appropriate conditions.
  • the conditions for growth of the host cell and expression of the chimeric protein will vary depending on various factors such as the host cell employed, the promoter and the particular chimeric protein being expressed. Those skilled in the art are capable of determining the appropriate conditions for the particular host/vector system employed. Methods for expressing a foreign gene in a host organism also are well known in the art (see, e.g., Maniatis et al. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 2 nd ed., 1989).
  • the gene encoding a particular polypeptide can be constructed by chemically synthesizing the entire nucleotide sequence, by amplification, such as by the polymerase chain reaction (PCR), or by cloning the gene of interest. The gene is then subcloned into an appropriate expression vector.
  • PCR polymerase chain reaction
  • Cloning vectors, expression vectors, plasmids, and viral vectors are well known in the art (see, e.g., Maniatis et al., supra, and Goedell, Methods in Enzymology, Vol. 185 (Academic Press 1990)).
  • Example 1 provides a detailed description of the preparation of a T7- based expression system useful for high-level expression of mammalian proteins in E. coli.
  • a Cys-His cleavage site within the sequence of the polypeptide by incorporating or mutating the appropriate nucleotides into the encoding nucleic acid by any of various methods including, for example, site-directed mutagenesis.
  • a Cys- His sequence can provide a site for cleavage by palladium complexes as described herein.
  • Recombinant methods can also be used to generate a nucleic acid encoding a protein with a repeating polypeptide sequence, each sequence separated by a Cys-His cleavage site. In this case, palladium complex-promoted cleavage can occur at multiple Cys-His sites in the polypeptide, releasing multiple copies of the desired peptide.
  • a Cys or a His residue may be incorporated into a site adjacent to the residue present in a peptide to create a Cys-His cleavage site, e.g., by site-specific mutagenesis.
  • selective cleavage can then be achieved at the incorporated Cys-His site to produce peptide fragments.
  • site-specific mutagenesis could be used to alter either the Cys or the His residue to another amino acid to prevent cleavage.
  • the process of the instant invention provides for the cleavage of chimeric proteins comprising peptides which include, but are not limited to, the glucagon-like peptide- 1 (GLP-1), glucagon-like peptide-2 (GLP-2), parathyroid hormone (PTH), parathyroid hormone related protein, growth hormone releasing hormone (GRF), adrenocorticotropic hormone (ACTH), enkephalins, endorphins, exendins, amylins, various opioid peptides, frog skin antibiotic peptides, such as gaegurins 5 and 6, brevinin 1, the ranatuerins 1 through 9, and the esculetins, glucose-dependent insulinotropic polypeptide (GIP), glucagon, motilin, thymopoietins, thymosins, ubiquitin, serum thymic factor, thymic humoral factor, neurotensin, tuftsin, and fragments and derivatives
  • polypeptides have an amide at their C-terminal and/or an -S-S- linkage in the molecule.
  • the precursor non- amidated or reduced forms of these peptides, respectively, can be expressed as a fusion construct with a Cys-His cleavage site incorporated and subjected to cleavage by palladium complexes in accordance with the process of the instant invention.
  • the product can then be amidated or oxidized to produce the final desired molecule.
  • leader sequences which can be employed with chimeric proteins include a signal sequence such as that used to direct secretion of a protein from a cell, the N-terminal portion of a mature protein sequence, such as from a structural gene, a linker sequence, or combinations thereof.
  • the chimeric protein has a molecular weight of between about 400 to about 100,000 daltons, or more preferably between 1,000 and 50,000 daltons and can comprise any of the natural amino acids, such as Ala (A), Arg (R), Asp (D), Asn (N), Glu (E), Gin (Q), Gly (G), His (H), Leu (L), He (I), Lys (K), Met (M), Cys (C), Phe (F), Pro (P), Ser (S), Thr (T), Tip (W), Tyr (Y), Val (V) (single letter amino acid code in parentheses), or may comprise any side chain-modified amino acid derivative commonly used in peptide chemistry.
  • the latter amino acid derivatives include, for example, nipecotic acid, 1- or 2-napthylalanines and p-benzoylamino-L- phenylalanine, among others.
  • Inclusion bodies can be recovered from the host cells by known methods such as, for example, lysing the cells chemically or mechanically and separating the inclusion bodies (chimeric protein) by centrifugation.
  • Cleavage is usually carried out at a temperature of between about 50 °C to about 70 °C. It is understood that the reaction conditions of the cleavage step of the process of the instant invention are adjusted depending on the palladium complex used and the characteristics of the polypeptide to be cleaved. The palladium complex should be solubihzed, which will affect the reaction conditions. Moreover, in a preferred embodiment, the reaction conditions used will at least partially denature the polypeptide to be cleaved.
  • Palladium (Pd) (II) complexes that can promote the cleavage of polypeptides in accordance with the instant invention include [Pd(OH 2 ) 3 (OH)] + , [PdCL , cis-[Pd(en)(OH 2 ) 2 ] 2+ , cis-[Pd(pn)(OH 2 ) 2 ] 2+ , cis-[Pd(pic)(OH 2 ) 2 ] 2+ , cis- [Pd(bpy)(OH 2 ) 2 ] 2+ , cis-[Pd(phen)(OH 2 ) 2 ] 2+ , and cis-[Pd(dtco-OH)(OH 2 ) 2 ] 2+ .
  • Pd (IV) complexed with chloride ion as hexachloropalladate can also provide an effective cleavage agent.
  • Palladium complexes can be prepared by methods well known in the art (see e.g., (H. Hohmann et al., friorg. Chirn. Acta, 174: 87 (1990); T. Rau et al, Inorg. Chem.. 36: 1454 (1997); C. Drexler et al., friorg. Chem.. 30: 1297 (1991), or U.S. patent no. 5,352,771) or can be purchased commercially.
  • Preferred palladium complexes include salts of the following: [PdC-U , [Pd(NCCH 3 ) 2 (OH 2 ) 2 ] 2+ > [PdCl 6 ] 2" , [Pd(dppe)(OH 2 ) 2 ] 2+ > [Pd(tpp)(OH 2 ) 2 ] 2+ , and [Pd(dppf)(OH 2 ) 2 ] 2+ .
  • Palladium complexes most preferred include [PdCl 4 ] 2 ⁇ [Pd(NCCH 3 )(OH 2 ) 2 ] 2+ , and [PdCl 6 ] 2" .
  • Complexes are used as the salt of an inorganic base, such as sodium or potassium. The sodium and potassium salts of [PdCLi] 2" are preferred . Selected examples of the coordination structures of palladium complexes are shown in Figure 1.
  • cleavage of the chimeric protein at the Cys-His site occurs at the carboxamide bond between the histidine residue and the residue immediately adjacent to the histidine on the C-terminal side.
  • the peptide NH 2 -Ala-Ala-Cys-His-Gly-Gly-Gly-COOH (SEQ ID NO:7) would be cleaved as follows:
  • the cleaved peptide produced in accordance with the process of the instant invention can be recovered from its fusion partner by ultrafiltration, precipitation, or more preferably, by reverse phase chromatography. Any commercially available reverse phase column suitable for the peptide being isolated may be employed. In many cases, the peptide recovered from the reverse phase column will refold into its native conformation, however, additional steps (e.g., oxidation) may be required to restore the peptide to a biologically active form, particularly when the peptide requires the formation of internal disulfide bonds for activity.
  • additional steps e.g., oxidation
  • Such steps can include, for example, HPLC, such as RP-HPLC or ion exchange chromatography steps.
  • the chimeric proteins T7tag-Ng-D 4 KCH-GRF (1-44)- Ala (SEQ ID ⁇ O:2), T7tag-Vg-D 4 K- CH-GRF(l-44)-CH (SEQ LD NO:6) and T7tag-Vg-G 5 PR-CH-PTH (1-34) (SEQ ID NO:4) were expressed in E. coli and were thereafter isolated in the form of an inclusion bodies.
  • This chimeric protem has a leader sequence connected by a cystinyl (Cys)-histidine (His) sequence to the growth hormone releasing factor peptide (GRF(1-44)CH).
  • T7tag-Vg-D 4 K-CH-GRF(l-44)-CH (SEQ ID NO:6), comprised a 14- residue signal sequence followed by both a 27 residue vestigial (Vg) sequence (which induced inclusion body formation and high expression) and a 13-residue linker which contained the Cys-His cleavage site.
  • the T7tag- Vg-D 4 K-CH-GRF(l-44)-Ala (SEQ ID NO:2) has a similar composition except that on palladium complex promoted cleavage the product is the growth hormone releasing factor analog GRF-Ala and finally, the T7tag-Vg-G 5 PR-CH- PTH(1 -34) (SEQ LD NO:4) is of similar construction and palladium promoted cleavage provides PTH(l-34), the natural hormone.
  • the isolated inclusion bodies were subjected to palladium-promoted acidic cleavage by (i) solubilizing them in a reaction mixture comprised of 80 % formic acid, and (ii) addition of tetrachloropalladate in a 10:1 molar ratio of chimeric protein (inclusion body) to tetrachloropalladate.
  • the cleavage reactions proceeded for between 1 to 24 hours, typically for about 1 to 6 hours, at a temperature ranging from about 50 °C to about 70 °C.
  • ethylenediamine, picolyl amine (2-aminomethyl pyridine or "pic"); methionine, or histidine were added to the reaction mixture to increase yield.
  • yields of around 75 % of substantially formylated peptide were obtained in around two hours using formic acid; around 95 % of this formylated peptide was deformylated in accordance with the process of the instant invention by removing the formic acid, and incubating the formylated peptide in 150 mM glycine and 25 mM hydroxylamine at pH 8.5 or incubating the formylated peptide in dilute acid for up to 16 hours.
  • the reaction mixture in formic acid was diluted up to 10 fold with dilute acid and held at about 15 to 30 °C for up to 16 hours, to achieve deformylation of the formylated peptide.
  • Palladmm(II) complexes were prepared as follows. Na PdCl , cis- [Pd(en)Cl 2 ], cz-?-[Pd(bp)Cl 2 ] and cts , -[Pd(phen)Cl 2 ] were purchased from Aldrich Chemical Co. and Strem Chemical Co. c ⁇ -[Pd(pn)Cl 2 ] (H. Hohmann et al.,
  • the complex [Pd(OH 2 ) 3 (OH)](NO 3 ) in solution was obtained by stirring a mixture of K 2 PdCl 4 and 3.98 equivalents of AgNO 3 for 2 hr at 50 °C.
  • Solid samples of palladium (II) or (IV) complexes were solubihzed in an appropriate aqueous solvent.
  • the pH of the reaction following addition of the palladium complex was adjusted to 1.5 or 1.74, as indicated, by addition of trifluoroacetic or hydrochloric acid.
  • the aqua complexes in solution were characterized by UV/Vis spectroscopy.
  • CH-GRF( 1-44)- Ala polypeptide were grown in 500 mL shake flasks containing tryptone, yeast, glucose, batch salts (sodium and potassium mono- and diphosphate salts and ammonium sulfate), and antibiotic. Inoculated shake flasks were subject to orbital shaking (200 rpm, 37 °C). Incubation was completed when the culture reached an optical density (OD) of 0.8-1.8 at 540 nm.
  • OD optical density
  • Fermentors ranging from 5 L to 100 L production capacities were seeded using shake flask cultures.
  • the media included batch salts, glucose, and chelated metals solution (potassium citrate, sodium citrate, magnesium sulfate, phosphoric acid ferric chloride, zinc chloride, cobalt chloride, sodium molybdate, manganese chloride, calcium chloride, and copper sulfate).
  • the pH of the medium was adjusted to 6.9 prior to inoculation and the pH was maintained at 6.9 during culture.
  • Dissolved oxygen was maintained at approximately 40 %, via agitation and supplemental oxygen.
  • Either silicone- based or polypropylene glycol-based "antifoam" was added aseptically on an "as needed" basis to reduce foaming in the fermentation culture.
  • Production fermentor cells were harvested from the fermentation broth by centrifugation.
  • Cell pellets were pooled, resuspended in an appropriate volume (for example, 2 L lysis buffer for material from a 5 L fermentor) of lysis buffer (6 g Tris free base and 0.93 g EDTA in 993 g water), and lysed in a high- pressure homogenizer.
  • Cell solids and peptide precursor-containing inclusion bodies were pelleted by centrifugation, collected, and dissolved via homogenization in 1.5 M citric acid / 1.0 mM EDTA solution, pH approximately 1.0 (for example, 1.5 L solution for material from a 5 L fermentor).
  • the pH of this suspension was adjusted to 4.9 by slow addition of 10 M sodium hydroxide with continuous mixing, while maintaining the temperature at less than 15 °C.
  • the precipitate containing the GRF precursor peptide was collected by centrifugation and washed twice by resuspending in water and centrifuging to a pellet.
  • Cleavage reactions were performed at a 10:1 molar ratio of palladium complex to solubihzed peptide. In general, cleavage reactions can be performed at ratios of about 1:1 to about 20:1 (palladium complex :peptide).
  • the purified peptide precursor precipitate (described above) was added to a solution of between 60 to 80 % formic acid, at a ratio of 1 g of paste per 50 mL formic acid, and homogenized under high shear. The solution was transferred into an appropriately sized reaction vessel and immediately blanketed with argon gas.
  • the solution was heated to 60 °C for approximately 1 hr and an aqueous solution of sodium tetrachloropalladate (6 mM) was added to achieve a 10: 1 molar ratio of palladium complex to peptide.
  • the reaction solution was incubated with stirring at 60 °C for 2 hr to effect cleavage of GRF(l-44)-Ala from the chimeric protein.
  • the reaction solution was cooled to 20 °C and diluted to 6 fold of the original volume with cleavage diluent (0.6 M NaCl, 10 mM sodium thiocyanate) and incubated at ambient temperature under argon for 30 min to stop the reaction and to allow precipitation of undesired peptides and remaining inclusion bodies.
  • the suspension was then filtered through a 0.45 ⁇ m filter. Reactions can also be stopped by methods well known in the art including, for example, column chromatography, evaporation, and freezing.
  • the filtrate from the above step was subjected to Arnberchrome CG-71 reversed-phase chromatography to separate cleaved GRF(l-44)-Ala peptide from leader and linker peptides, sodium thiocyanate, other contaminating proteins, residual palladium, and formic acid.
  • the crude GRF(l-44)-Ala solution was loaded at 12-35 mg peptide per mL resin onto an Amberchrome CG-71 column, previously equilibrated in Buffer A (5 % ethanol, 5 mM HC1), and washed with 7-11 column volumes of 5 mM HC1, to remove unbound material.
  • GRF( 1-44)- Ala peptide was then eluted with a linear gradient set from 0 to 100 % Buffer B (95 % ethanol, 5 mM HC1) over 3 column volumes. Collection of the GRF(l-44)-Ala peak started when the peptide absorbance (at 280 nm) began to rise; the peak of interest eluted as a split peak at about 40 % Buffer B and continued on the backside of the second peak until the absorbance leveled off.
  • Deformylation of formylated Ser and Thr residues in the crude GRF(1- 44)-Ala pool from the Amberchrome column was accomplished by diluting the GRF(l-44)-Ala solution with 3 volumes of aqueous 8 M urea; glycine was then added to 0.048 M and the pH was adjusted to pH 9.0 with NaOH. The mixture was stirred for 30 min at 23 °C. Following deformylation, the pH of the GRF(1- 44)-Ala peptide product solution was adjusted to 7.0 with HC1, and filtered through a 0.45 ⁇ m filter.
  • the buffer system was composed of Buffer A (5 % acetonitrile, 0.1 % TFA) and Buffer B (95 % acetonitrile, 0.1 % TFA). Eluted sample peaks were identified by absorbance at 214 nm. Test samples were run in triplicate, and the average area under the eluted product peak was compared to the peak area from a similarly detected triplicate series of a GRF reference concentration standard (1.00 ⁇ 0.25 mg/mL). The concentration of GRF(l-44)-Ala was then calculated by comparison with the peak area of the reference standard. A single major peptide peak was observed for the deformylated GRF(l-44)-Ala in the orthogonal HPLC system.
  • the deformylated GRF(l-44)-Ala cleavage product was transamidated in accordance with the process of U.S. Patent 5,580,751.
  • the resultant putative GRF(l-44)amide was sequenced by Edman degradation and was found to have the amino acid sequence expected for GRF(l-44)amide, thereby confirming that deformylation was essentially complete.
  • rGRF-CH Expression of the T7tag-Vg-D 4 K-CH-GRF(1-44VCH (SEO ID NO:6)
  • T 7 tag-Vg-D 4 KCH-GRF (l-44)-CH (SEQ ID NO:6) ( Figure 6) was recombinantly expressed in E. coli in accordance with techniques analogous to those disclosed for the expression of rGRF-Ala in Example 1. 2. Solubilization And Cleavage Of T7tag-Vg-D 4 K-CH-GRF(1-44VCH
  • the Microsorb-MV CNC 8 reverse-phase HPLC method used a Microsorb-MV Cyano-C 8 100 A, 5 ⁇ m, 4.6 x 150 mm column.
  • the gradient for analysis of the quenched palladium reactions was as follows: 20 - 30 %B (3 min.), 30 - 40 %B (6 min.), 40 - 100 %B (1.5 min), 100 - 20 %B (0.6 min.), 20 %B (3 min.) at 1 mL/min at 30 °C.
  • the Waters Symmetry C 18 reverse-phase HPLC method (or Waters Symmetry C 18 reverse-phase HPLC method) used a Waters Symmetry C 18 , 100 A, 3.5 ⁇ m, 4.6 x 150 mm column (Catalog #Wat200632).
  • the gradient was 25 - 33 %B (24 min.), 33 - 60 %B (6 min.), 60 - 90 %B (1 min.), 90 - 25 %B (0.5 min.), 25 %B (4 min.) at 1 mL/min and 40 °C.
  • the absorbance was monitored from 210 - 320 nm.
  • the LC/MS method utilized a Waters Symmetry C 18 column, 100 A, 3.55 ⁇ m, 2.1 x 150 mm column (Catalog #Watl06005).
  • the gradient was 20 - 30 %B (1 min.), 30 - 50 %B (25 min.), 50 - 100 %B (4 min.), 100 - 20 %B (1 min.), 20 %B (7 min.) at 0.25 mL/min at 40 °C.
  • the absorbance was monitored from 210 - 320 nm.
  • the LCQ Duo MS detector was set to source from 7 — 19 min. and 24 - 30 min.
  • the scan event was set from 700 - 2000 m/z units.
  • a treatment method for preparing the samples referred to as the diethyldithiocarbamate(ddtc) treatment, was developed. The treatment was as follows: 50 ⁇ L of sample was added to 425 ⁇ L of 20 mM ddtc in 8 M urea and allowed to stand at room temperature for five minutes.
  • Tetrachloropalladate at 4.5, 6.75 and 9 mM was used and the cleavage monitored by HPLC with time points taken every hour. The reactions were done at 60 °C. The time points were diluted six-fold with 48 mM thiourea (40 mM final thiourea). All analytical analyses were done as described using the Microsorb-MV CNC 8 reverse-phase HPLC method. The results are summarized in Figure 2. Yields are around 40 % in 60 % formic with a maximum in one hour but in 80 % formic acid yields as high as 75 % are achieved. Over the range of tetrachloropalladate concentrations employed (4.5 to 9 mM) there is no appreciable difference in yield.
  • Tetrachloropalladate promoted cleavage in 75 % formic acid was investigated over an hour period at 60 °C.
  • the precursor peptide T7tag-Vg- D 4 K-CH-GRF(l-44)-CH (SEQ ID NO:6) was solubihzed into 78 % formic acid and diluted to 75 % for the reaction. Palladium was added to 4.5 mM from a stock solution. The reaction was at 60 °C for one hour, with time points taken at the following times: 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, and 60 minutes. Each time point was diluted six-fold with 48 mM thiourea.
  • Figure 3 shows a plot of the time course of this reaction showing that in about 60 minutes under these conditions up to 80 % cleavage to GRF-CH is obtained. As shown the reactions were done in duplicate and the average yield for the two reactions is plotted as the line. This shows that the cleavage is efficient, rapid and reproducible.
  • Thiourea is a good chelating reagent to palladium (The Chemistry of Platinum and Palladium, F.R. Hartley, Applied Science, 1993, Publishers LTD, London, 1993). Both ethylenediamine and thiourea were added as ligands during the palladium cleavage in 80 % formic acid to assess if they might improve the reaction speed or yield by preventing reduction of palladate that accompanies reactions in formic acid. The reactions were prepared and performed using the aforementioned conditions, except that thiourea was added in a 1 : 1 ratio with palladium in one reaction and ethylenediamine was added in a 2:1 ratio with palladium in another reaction. Each ligand was added from a stock solution.
  • DNA encoding PTH(l-34) was cloned downstream of a 10-amino acid leader sequence in a bacterial expression vector to generate T7tag-Vg-G 5 PR-CH- PTH (1-34) ( Figure 5) (SEQ ID NO:4).
  • the expressed chimeric protein is 91 amino acids in length.
  • Inclusion body preparations were obtained from E. coli tranformed with the expression construct essentially as described above for the production of GRF(1-44)CH. Precipitated inclusion bodies were washed by resuspension in water with sonication, centrifuged to obtain a pellet, then dissolved at a concentration of 6 mg/mL in the medium and palladium complex mixtures, as indicted in Table 1.
  • EXAMPLE 7 Tetrachloropalladate promoted hydrolytic cleavage of oxidized T7tag-Vg-D K- CH-GRF(l-44)-A (SEO LD NO:2) in formic acid.
  • An aged sample of T7tag-Vg-D 4 K-CH-GRF(l-44)-A (SEQ ID NO:2) was shown to be oxidized to (T7tag-Vg-D 4 K-CH-GRF(l-44)-A (SEQ ID NO:2)) 2 by non-reducing gel electrophoresis.
  • the dimer was in roughly a 2: 1 ratio compared to the monomer.
  • the mixture was treated with tetrachloropalladate in 80 % formic acid for 2 hours at 60 °C.
  • the reaction solution was diluted to 10 fold with 25 mM thiourea and 8 M urea followed by centrifugation. The resulting supernatant was analyzed on non-reducing protein gel electrophoresis. The peptide dimer band had disappeared and only a trace amount of unreacted monomer was still observed on the gel. The cleavage yield was about 60 %. This experiment shows that even if the Cys residue of the Cys- His cleavage site is in the disulfide state cleavage still occurs.

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1532262A2 (de) * 2002-05-24 2005-05-25 Restoragen, Inc. Peptidamidierungsverfahren
US7592432B2 (en) 2002-05-24 2009-09-22 Restoragen, Inc. Polypeptide cleavage process
US7781567B2 (en) 2002-05-24 2010-08-24 Nps Pharmaceuticals, Inc. Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides
US7829307B2 (en) 2003-11-21 2010-11-09 Nps Pharmaceuticals, Inc. Production of glucagon-like peptide 2

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AU2003237244B2 (en) * 2002-05-24 2009-11-12 Medtronic, Inc. Polypeptide cleavage process

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1194795B (it) * 1981-05-13 1988-09-28 Pierrel Spa Procedimento per la eliminazione del gruppo n-formile da peptidi n-formilati e da esteri di peptidi n-formilati
IL84110A (en) * 1986-10-14 1992-11-15 Lilly Co Eli Process for transforming a human insulin precursor to a human insulin
US5580751A (en) * 1990-09-14 1996-12-03 Carlsberg A/S Process for the preparation of C-terminally amidated peptides
US5352771A (en) * 1992-08-31 1994-10-04 Iowa State University Research Foundation Inc. Hydrolysis of peptide bonds using Pt (II) and Pd (II) complexes
US5393924A (en) * 1992-10-02 1995-02-28 Hoechst Celanese Corporation Method for the preparation of arylalkanolamineacylates
US6376529B1 (en) * 1995-06-07 2002-04-23 Peng Cho Tang Mono- and bis-indolylquinones and prophylactic and therapeutic uses thereof
DE69726868T2 (de) * 1996-07-19 2004-10-28 Monsanto Technology Llc. DEFORMYLIERUNG VON f-MET PEPTIDEN IN BAKTERIEN EXPRESSIONSSYSTEMEN
US6660758B1 (en) * 1996-12-13 2003-12-09 The Scripps Research Institute Epothilone analogs
DE60334912D1 (de) 2002-05-24 2010-12-23 Medtronic Inc Polypeptid-spaltungsprozess
WO2003099853A2 (en) * 2002-05-24 2003-12-04 Restoragen, Inc. Petide amidation process
AU2003237244B2 (en) * 2002-05-24 2009-11-12 Medtronic, Inc. Polypeptide cleavage process

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DOU ET AL.: "Preliminary Study On The Cleavage Of Fusion protein GST-CMIV With Palladium(II) complex", PREP. BIOCHEM & BIOTECHNOL., vol. 30, no. 1, 2000, pages 69 - 78, XP002438893

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1532262A2 (de) * 2002-05-24 2005-05-25 Restoragen, Inc. Peptidamidierungsverfahren
EP1532262A4 (de) * 2002-05-24 2009-06-10 Restoragen Inc Peptidamidierungsverfahren
US7592432B2 (en) 2002-05-24 2009-09-22 Restoragen, Inc. Polypeptide cleavage process
US7781567B2 (en) 2002-05-24 2010-08-24 Nps Pharmaceuticals, Inc. Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides
US8148508B2 (en) 2002-05-24 2012-04-03 Nps Pharmaceuticals, Inc. Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides
US8153762B2 (en) 2002-05-24 2012-04-10 Medtronic, Inc. Composition for palladium-mediated cleavage of peptides containing CXC, CXH or CHM sequences
US7829307B2 (en) 2003-11-21 2010-11-09 Nps Pharmaceuticals, Inc. Production of glucagon-like peptide 2

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